Characterization of the overall topology and inter-subunit contacts of protein complexes, and their assembly/disassembly and unfolding pathways, is critical because these complexes regulate key biological processes, including processes where malfunction leads to disease. Although many protein complexes can be characterized by existing tools, including X-ray crystallography and NMR, others are not amenable to these approaches. One solution to this gap in the field is addition of native mass spectrometry (MS) tools to the suite of structural biology approaches. While native MS has provided many useful insights on protein complexes, the most common activation method, collision-induced dissociation (CID, multiple low-energy collisions with gaseous targets to induce fragmentation), often induces an unfolding pathway that produces highly charged unfolded monomers and complementary (n-1)-mers,. This information is insufficient to directly characterize the topology and intersubunit contacts of the complexes. Surface-induced dissociation (SID), in contrast, has been shown by the PI's lab to induce direct dissociation to subcomplexes and to also effectively probe subtle structural changes that are not evident from CID spectra (e.g. pre-unfolding in the ion source). The overarching goal of this proposal is to develop this novel, alternative MS activation method, surface-induced dissociation coupled to ion mobility (SID/IM), as a tool for characterization of structural features of multimeric protein complexes so that these features can be tied to function. Aim I is to characterize pentraxin-ligand structures of increasingly highe complexity, including binding of metal ions plus small molecular ligands and protein ligands to C-reactive protein and PTX3. Aim II will determine whether SID/IM can define the structural features and unfolding propensity of ROP (repressor of primer) dimer mutants and ROP mutant:RNA complexes. Aim III will determine whether SID/IM can define whether discrete modifications of histone proteins influences nucleosome (histone:DNA) fragmentation/disassembly in ways related to their expected destabilization. Aim IV will utilize SID/IM to characterize a lipid:protein complex (CRP:DMPC), membrane receptor:protein complex (CTB:GM1) and membrane proteins (rhodopsin, aquaporin0, YidC, YidC:Pf3 coat) sprayed from detergent (e.g., DDM), amphipols, or nanodiscs. Multiple collaborators, experts in the production/purification/functional behavior of particular protein complexes but who need access to better characterization tools, have agreed to provide proteins of varying complexity (Bortazzi (Instituto Clinico Humanitas; PTX3); Magliery (OSU; ROP mutants); Poirier and Ottesen (OSU; modified nucleosomes); Brown (UA; rhodopsin); Schey (Vanderbilt; AQP-0); Dalbey (OSU; YidC, Pf3 Coat). Characterization of the innovative SID/IM approach, direct comparison with CID/IM, and development of knowledge of sample types where the SID approach is vital to protein complex structure determination, is critical for the continued development of native MS as a strong structural biology tool.